This application claims the benefit of Korean Patent Application No. 10-2004-0007528, filed on Feb. 5, 2004, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a microorganism having an inactivated tyrR gene, a method of producing the same and a method of producing L-threonine using the microorganism.
2. Description of the Related Art
L-threonine is an essential amino acid and is widely used as a feed and food additive, and also as a pharmaceutical and raw material for synthesizing some drugs. It has been produced by fermentation with artificial mutants of the genus Escherichia, Coryneform bacteria, Seratia and Providencia. For example, Japanese Patent Publication No. 10037/81 discloses a method of producing L-threonine using a strain belonging to the genus Escherichia which has a requirement for diaminopimelic acid and methionine, and has the resistance to the feedback inhibition by threonine of the biosynthetic system of threonine. Japanese Patent Application Laid-open No. 224684/83 discloses a method of producing L-threonine using a strain belonging to the genus Brevibacterium which is resistant to S-(2-aminoethyl)-L-cysteine and α-amino-β-hydroxy valeric acid and has a nutritional requirement for L-isoleucine and L-lysine. Korean Patent Application Laid-open No. 8022/87 discloses a method of producing L-threonine using a diaminopimelic acid and methionine-requiring, α-amino-β-hydroxy valeric acid-resistant strain belonging to the genus Escherichia which has an additional resistance to at least one substance selected from the group consisting of rifampicin, lysine, methionine, aspartic acid, and homoserine, or has a reduced ability to decompose L-threonine. Japanese Patent Application Laid-open No. 219582/90 discloses a method for producing L-threonine using a strain belonging to the genus Providencia which is resistant to α-amino-β-hydroxy valeric acid, L-ethionine, thiaisoleucine, oxythiamine, and sulfaguanidine, and has a requirement for L-leucine and also a leaky requirement for L-isoleucine.
However, the above known methods have the disadvantages that they fail to afford a high production of L-threonine or require costly requirements such as diaminopimelic acid and isoleucine. In other words, the use of diaminopimelic acid-requiring strains in the production of L-threonine includes an additional fermentation of diaminopimelic acid and thus may increase cost. Where a strain having a requirement for isoleucine is used for the production of L-threonine, costly isoleucine must be added to fermentation media, which increases cost.
In an attempt to overcome these disadvantages, the present inventors developed an L-threonine-producing strain of Escherichia coli which is resistant to L-methionine, L-threonine and L-lysine analogues and α-aminobutyric acid, and has a nutritional requirement for methionine and a leaky requirement for isoleucine. They successfully produced L-threonine by fermentation with the strain at higher yields than with prior strains. The strain and a method for producing L-threonine using said strain are disclosed in Korean Patent Publication No. 92-8365.
Oxidative decomposition of tyrosine has two routes, i.e. decomposition to fumarate and acetoacetate and decomposition after conversion into 3,4-hydroxyphenylacetate by tyrosinase. In the former case, L-tyrosine reacts with α-ketoglutarate to produce L-glutamate and 4-hydroxyphenylpyruvate (Neidhardt FC et al., (eds) Escherichia coli and Salmonella: cellular and molecular biology, 2nd edn. ASM Press. Washington D.C., 1996, pp. 2649-2660). The biosynthesis of tyrosine and phenylalanine includes transamination. An important enzyme in the transamination is encoded by tyrB, aspC and ilvE genes (Ji Yang and James Pittard, Journal of Bateriology, 1987, 167, pp. 4710-4715). That is, the tyrB gene encodes a subunit of aromatic amino acid transaminase. A TyrB protein encoded by the tyrB gene is involved in the transamination by the aromatic amino acid transaminase in the third and final steps of tyrosine biosynthesis as the subunit (Neidhardt FC et al., (eds) Escherichia coli and Salmonella: cellular and molecular biology, 2nd edn. ASM Press. Washington D.C., 1996, pp. 2649-2660). The tyr B gene also has a function similar to aspC gene to be involved in the synthesis of aspartate (ASP) from oxaloacetate (OAA) as well as the biosynthesis of tyrosine and phenylalanine. ASP is an intermediate for L-threonine biosynthesis (Neidhardt FC et al., (eds) Escherichia coli and Salmonella: cellular and molecular biology, 2nd edn. ASM Press. Washington D.C., 1996, pp. 358-403).
The tyrB gene belongs to tyrR regulon and undergoes repression of expression during trasncription by a product encoded by tyrR gene (Ji Yang and James Pittard, Journal of Bateriology, 1987, 167, pp. 4710-4715).
The present inventors have intensively studied to select strains having an improved ability to produce L-threonine on the basis of conventional technologies and now discovered that L-threonine biosynthesis can be facilitated by inactivation of the tyrR gene.